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FLAVONOID GLYCOSIDES FROM BYRSOCARPUS COCCINEUS LEAVES. SCHUM AND THONN (CONNARACEAE) A. A. Ahmadu*1, H. S. Hassan1 ,M. U Abubakar1 and I. N. Akpulu2
1
Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello, University, Zaria-Nigeria., 2 Department of Pharmaceutics and Pharm. Microbiology, Ahmadu Bello University, Zaria-Nigeria
*E-Mail: ahmadu2001@yahoo.com
Abstract
The bioactive ethyl acetate and N-butanol soluble parts of an ethanolic extract of Byrsocarpus coccineus leaves was subjected to column chromatography over silica gel G (60 - 120) and repeated purification of the flavonoid rich fraction over sephadex LH-20 eluted with methanol led to the isolation of three flavonoid glycosides identified as quercetin 3-O--arabinoside (I), quercetin(II) and quercetin 3--D-glucoside. Their structures were elucidated by 1H and 13C-NMR data and are reported here for the first time in this plant. Key words: Flavonoids Quercetin 3-O--arabinoside,Quercetin,Quercetin 3-O--D-glucoside
Introduction
The plant Byrsocarpus coccineus is indigenous to Africa especially Togo, Ghana and Nigeria (Dalziel, 1955). Folkloric uses of the plant include: The leaf decoction for venereal diseases and as antidote to arrow poisoning in Ghana; decoction of the leaves is applied to sore of mouth and skin, the Yorubas of Western Nigeria use the cold infusion of the bruised leaves for gonorrhoea, and the plant is considered as a urinary sedative (Irvine,1961). A decoction of the leaves is drunk as remedy for pile, while the decoction of the whole plant is applied to swelling and tumours and also to arrest bleeding, the plant has also been reported as a remedy for diarrhoea (Akindele and Adeyemi, 2006). The uterotonic and molluscidal activities of the ethanol extract have been reported (Amos et al, 2002; Okunji and Iwu,1988). We have found the antibacterial activity of the ethyl acetate and N-butanol soluble parts of the crude ethanol extract (Ahmadu et al, unpublished).A coumaroyl derivative have been isolated from the leaves of this plant Vickery and Vickery, (1980). To our knowledge, there had been no documented evidence in the isolation of flavonoids from this plant. In our continuous search for bioactive flavonoids from our medicinal plant, we report here the isolation of three flavonoids identified as quercetin 3-O--arabinoside, quercetin and quercetin 3-O--D-glucoside from the bioactive ethyl acetate and n-butanol soluble parts of ethanolic extract of Byrsocarpus coccineus.
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Extraction and Isolation The air-dried powdered leaves (220g) were extracted exhaustively with petroleum ether (60 800C) using maceration. The combined extract was concentrated in vacuo to give a dark-green mass 6.5g (2.95%w/w). The marc (200g) was extracted to exhaustion with 95% ethanol to give a greenish mass 27.6g (12.95%w/w). 20g of the crude ethanolic extract was suspended in water and successively extracted with ethyl acetate (2 x 500ml) and N-butanol (3 x 500ml) and evaporation of the solvents at reduced pressure gave 1.4g of ethyl acetate (brownish - mass) and 1g of N-butanol (reddish-brown mass). 1.3g of the ethyl acetate soluble part was packed with 50g of silica gel G in a column (75cm x 2.0cm) and eluted gradiently with chloroform; chloroform: methanol mixtures 9: 1, 8: 2, 7: 3 and methanol. The progress of separation was monitored by thin layer chromatography using solvent system I: ethyl acetate : methanol : water 100 : 16.5 : 13.5 and solvent system II : ethyl acetate : formic acid : water 10 : 2 : 3. Fractions eluted with chloroform: methanol (9: 1) homogeneous on TLC afforded the flavonoid mixture (52mg). This was subjected to repeated column chromatography (25cm x 1.1cm) over sephadex LH-20 eluted with methanol to afford a yellowish-brown powder 8mg, mp 235-237oC. TLC using solvent system I, and II gave a single spot with Rf (0.61, and 0.72) respectively. Compound I was identified by 1H-NMR (ID&2D), and 13C-NMR (ID&2D), HSQC, DEPT. The same procedure was repeated for n-butanol soluble part (1g) to give compound II as a yellowish powder (5mg) mp 162164oC and III (3.5mg) identified as quercetin and quercetin 3-O--D-glucoside respectively by direct comparison of the 1H-NMR with that reported in literature (Mabry and Markham, 1968, Mabry et al, 1970). Table 1: 1H-NMR of Compound I (Lozoya et al.,1994,Markham et al.,1978) Position DEPT aH bC C/H 2 C 158.7 3 C 135.7 4 C 179.5 5 C 163.2 6 CH 6.20(d, 1.6Hz) 100.1 7 C 166.8 8 CH 6.39(d, 1.2Hz) 94.90 9 C 158.60 10 C 105.7 1/ C 123.0 2/ CH 7.74(d, 1.5Hz) 117.6 3/ C 146.1 4/ C 150.1 5/ CH 6.88(d, 8.5Hz) 116.3 6/ CH 7.58(d,d, 2.0Hz, 8.0Hz) 123.16 -arabinose HO 1// CH 5.17 (d, 3.5Hz) 104.8 2// CH 73.02 3// CH 69.29 4// CH 3.65 3.62(d,d, 1.5, 7Hz) 74.28 OH 5// CH2 3.46 3.48 (d, J = 7.0Hz) 67.1 COMPOUND
OH OH O
OR O
R - arabinose H -D-glucose
I II III
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3.64d,d(1,7Hz)
Acknowledgement
The authors wish to acknowledge the assistance of Dr. Simon Gibbons of the Centre for Phytotherapy Research, University of London for the NMR analysis.
References
1. Akindele, A. J. and Adeyemi, O. O. (2006) Evaluation of antidiarrhoeal activity of Byrsocarpus coccineus. J. Ethnopharmacol. 108(1): 20-25 2. Amos, S; L. Binda, Kunle, O.F; C. Wambebe and K. Gamaniel (2002). Uterotonic Properties of the ethanol extract of Byrsocarpus coccineus. Pharml Biol 40 (1):33 38. 3. Dalziel, J. M. (1955). The useful plants of West Tropical Africa. A Crown Agent for Oversea Publication. London pp568 570 4. Irvine, F. R. (1961). Woody Plants of Ghana, An Oxford University Press Publication, London. PP568 570
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5. Lozoya, X., Meckes, M., Abou-Zaid, M., Turtoiello, J., Nozzolillio, C. and Arnson, ,J. (1994). Quercetin glycosides in Psidium guajava L. leaves and determination of spasmolytic principle. Arch Med Res. 25(1): 11-16 6. Mabry, T. J., Markham, K. R and Thomas, M. B. (1970). The Systematic Identification of Flavonoids. SpringerVerlag Publication, New York. 261-266,294 7. Mabry, T. J. and Markham, K. R. (1968). Flavonoids from Bauhinia species. Tetrahedron 24: 823-826. 8. Markham, K R., Ternai, B., Stanley, R., Geiger, H and Mabry, T. J. (1978). 13C-NMR Studies of Flavonoids II. Tetrahedron 34: 1391-1397 9. Okunji, C. O and Iwu, M. M. (1988).Control of Schistosomiasis using Nigerian Medicinal plants as molluscicides. Int. J. crude Drug Res. 26(4): 246 252. 10. Vickery, M and Vickery, B. (1980). Coumarins and related compounds in members of the Connaraceae. Toxicol. Letter: 5: 115 118 11. Young, H. C., Young, H.L., Hosup, Y and Jingwood, K. (1996). A Flavonoid diglycoside from Lepisorus ussuriensis Phytochemistry 43(5): 1111-1113.